Effect of sample size and staining methods on stallion M. HIDALGO

Original Paper
Vet. Med. – Czech, 50, 2005 (1): 24–32
Effect of sample size and staining methods on stallion
sperm morphometry by the Sperm Class Analyzer
Reproduction and Obstetrics Unit, Department of Animal Medicine and Surgery, University
of Cordoba, Cordoba, Spain
Physical Anthropology Unit, Department of Functional Biology and Physical Anthropology,
University of Valencia, Valencia, Spain
ABSTRACT: Computer-assisted sperm morphometry analysis has improved the assessment of sperm morphology,
but the results depend on the use of adequate evaluation and staining procedures of spermatozoa from individual
species. In this study, the morphological module of the Sperm Class Analyzer was used for the morphometric
analysis of stallion sperm heads and midpieces. Semen samples were obtained from six fertile stallions in order
to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris’ Haematoxylin) on the
accuracy of image processing and sperm morphometry, and the effect of the sample size on sperm morphometric
measurements. Harris’ Haematoxylin was the staining technique of choice on the accuracy of the image processing with an optimum contrast of sperm cells with the surrounding background that allows an efficient boundary detection and segmentation which results in the highest proportion of sperm heads and midpieces assessed
(80.47%). The results indicate that the staining methods affected significantly the sperm dimensions with increased
values from Diff-Quik than Hemacolor and Harris’ Haematoxylin respectively (Diff-Quik > Hemacolor > Harris’
Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa
were analysed. In conclusion, to obtain objective and accurate sperm morphometric measurements by the Sperm
Class Analyzer system in the stallion, it’s recommended the analysis of 100 spermatozoa from slides which have
been previously stained with Harris’ Haematoxylin.
Keywords: ASMA; sperm head; midpiece; morphometric anlaysis; validation
The evaluation of sperm quality is useful in predicting the fertility of sperm donors (Colenbrander
et al., 2003) and is of great importance in maximizing reproductive efficiency, either under natural
breeding conditions (Jasko et al., 1990) or in programs of assisted reproduction (Rodriguez et al.,
2001). Furthermore, it is a useful tool in the clinical diagnosis of subfertile animals. Conventional
evaluation techniques have been based on the
subjective assessment of semen parameters such
as motility, morphology and semen volume or concentration (Verstegen et al., 2002). Abnormalities
in sperm morphology are an important indicator of decreased fertility in humans (Kruger et al.,
1988) and some animal species (Voss et al., 1981;
Chandler et al., 1988; Sekoni and Gustafsson, 1987)
and have also been used as an indicator of the effect
of various toxicants on sperm production (Foote
et al., 1986).
However, the subjective assessment of sperm
morphology based on visual observation has led
to widely varying results due to numerous factors
such as the use of different staining procedures
or the experience of technicians, among others.
According to Zaini et al. (1985), the variability in
results can range from 40–60%, demonstrating
the low repeatability of these methods (Jequier
and Ukombe, 1983; Ombelet et al., 1997; Cooper
et al., 1999). These variations make it difficult to
accurately interpret data, underscoring the need
for techniques which are objective, precise and
Vet. Med. – Czech, 50, 2005 (1): 24–32
In the 1990’s, the introduction of automated
sperm morphometry analysis systems (ASMA)
attempted to overcome the problem of the subjectivity of visually based methods of assessment.
Although this technology was originally designed
for human sperm (Davis et al., 1992; Kruger et al.,
1993; de Monserrat et al., 1995), it has been progressively adapted to some animal species (Gravance
et al., 1996; Sancho et al., 1998; Iguer-Ouada and
Verstegen, 2001). These systems are capable of detecting subtle differences that conventional methods were unable to identify (Jagoe et al., 1987), such
as the relationship between sperm morphometry
and fertility (Casey et al., 1997).
For accurate sperm morphometry analysis, a
number of analytical variables are used for each
species. Currently, the precision of ASMA systems
depends upon the standardization of these variables (Davis and Gravance, 1993; Gago et al., 1998;
Gravance et al., 1995; Hidalgo et al., 2004), namely
appropriate sample preparation (washing, fixation
and staining) and correct microscopic image analysis. In addition to the variations inherent to the
evaluation process, errors are often the result of
differences between ASMA systems or the fact that
an insufficient number of spermatozoa are analysed
which are not representative of the sample.
The aims of the present study were to evaluate
the effect of three different staining procedures
on the accuracy of image processing and sperm
morphometry, and the effect of the number of
spermatozoa analysed to obtain a representative
assessment of a stallion semen sample for sperm
head and midpiece morphometry using the Sperm
Class Analyzer ASMA system.
Original Paper
were extended in a skim milk diluent and placed
in an incubator at 37ºC. Motility was evaluated
using the Sperm Class Analyzer  (SCA) motility
module (the features are described in the section
on morphometric analysis). Sperm concentration
was calculated with a haemocytometer and a slide
was prepared for subjective analysis of sperm morphology.
For morphometric analysis, 200 µl of the diluted sperm were deposited in the same volume of
Dulbecco’s Phosphate Buffered Saline (DPBS) in an
eppendorf tube and centrifuged at 600 g for 10 min
after removing the supernatant, the sperm pellets
were resuspended in DPBS to a concentration of
50 million sperm/ml. One drop of 7 µl of the final
dilution was placed on a microscopic slide and allowed to air dry.
Staining methods
Three semen smears per ejaculate and per stallion were stained with each of the three following
staining techniques: Diff-Quik (DQ) (Baxter DADE
AG 3186, Düdingen, Switzerland), Hemacolor (HC)
(Merck, Darmstadt, Germany, Cat. no. 11661) and
Harris’ Haematoxylin (HH) (Papanicolau solution 1a,
Merck Cat. no. 9253, Darmstad, Germany). Manufacturers’ instructions were followed for the first
and second method, although 1 and 2 minutes increased the time proposed for each step, respectively. The third semen smears were stained with
Harris’ Haematoxylin by leaving the slide in the
stain for 40 minutes.
Once stained, all the slides were identified and
permanently sealed with Eukitt mounting medium
(Kindler & Co, Freiburg, Germany) and a coverslip.
Semen collection and sample preparation
Morphometric analysis
Semen samples were collected from six adult
Spanish Thoroughbred stallions using an artificial
vagina (Missouri model). All stallions were actively
being used for natural service breeding with physiological fertility and semen parameters (motility,
sperm concentration and subjectively assessed
sperm morphology). One representative ejaculate
per stallion was assessed in the experimental design.
After semen was collected, the volume of each
gel-free ejaculate was recorded. The semen samples
Morphometric analysis of the sperm head and
midpiece was performed using the morphological module of the SCA version 2002 (Microptic
SL, Barcelona, Spain). The equipment consisted
of a microscope (Olympus BH-2; Tokyo, Japan)
equipped with a bright-field 100× objective and a
3.3× photo-ocular. A video camera (Sony CCD-IRIS
SSC-M370CE; Sony Corporation, Tokyo, Japan)
was mounted on the microscope to capture the
images and transmit them to the video digitizer
board (Meteor II; Matrox Electronic Systems Ltd,
Original Paper
Vet. Med. – Czech, 50, 2005 (1): 24–32
Quebec, Canada) located in a Pentium processor.
The SCA computer system included a high-resolution principal monitor (Sony Multiscan 200 SX;
Sony Corporation, Tokyo, Japan) and sperm image analysis software. The array size of the video
frame grabber was 512 × 512 × 8 bit providing digitised images of 262 144 pixels and 256 grey levels.
Resolution of images was 0.08 µm per pixel in the
horizontal and vertical axes.
The spermatozoa were captured randomly in different fields with a 100× oil immersion objective
rejecting only those that overlapped. This process
was performed manually by interactive selection
of cells to avoid the inclusion of foreign particles
that interfered in the way of the posterior image
processing (Figure 1a). The digitised cells were automatically segmented with a range of grey-level
values predetermined by the analysis factor (the
automatic algorithm to define the contrast between
cell and field). The system detected the boundary
of sperm heads and midpieces and their outlines
were displayed as white overlays superimposed on
the microscopic video image (Figure 1b). When
the boundary did not match the microscopic image
profile, the analysis factor was modified. When it
was not possible to obtain a correct boundary, the
cells were eliminated.
Thirteen morphometric parameters were calculated automatically: four for head size: length (L, in
µm), width (W, in µm), area (A, in µm2) and perimeter (P, in µm); four for head shape calculated from
the previous parameters: ellipticity (L/W), rugosity
(4πA/P2), elongation ((L – W)/(L + W)), regularity
(πLW/4A); and four for the midpiece: width (w,
in µm), area (a, in µm 2), distance (d, in µm) (between the major head axis and the midpiece) and
angle (o) (the angle of divergence of the midpiece
and the head axis) (Figure 2). The measurements of
each individual sperm cell were saved in an Excel
(Microsoft Corporation, Redmon, Washinton,
USA) compatible database by the software for further analysis.
Experimental design
Effect of the staining technique on the accuracy of image processing. In order to determine the
adequacy of the three staining techniques for capture and subsequent digitisation and binarization
of images, at least 100 spermatozoa from each slide,
1 per staining and per animal, were captured and
subsequently analysed totalising 3 700 spermatozoa
over the entire semen samples. The percentage of
sperm heads and midpieces which had been converted into correct binary images was determined
visually by checking if the boundary assigned by the
SCA to the spermatozoa matched its microscopic
image profile and correctly delineated the sperm
head and midpiece.
Effect of the staining technique on sperm morphometry. A minimum of 100 spermatozoa was
analysed per slide using each of the three staining
techniques, for all six animals. The morphometric
parameters obtained with each method were then
Effect of the number of spermatozoa analysed.
To determine the minimum sample size needed to
characterize the whole population, 200 sperm cells
were analysed on each of the slides stained with
HH for all six animals. Subsets of 100, 150, 175 and
200 spermatozoa were randomly selected from the
initial reference group of 200 and compared.
Statistical analysis
For each morphometric parameter, normality of
the data distributions and variance homogeneity
were checked by the test of Kolmogorov-Smirnov
Figure 1. Frame grabber and boundary of a sperm cell
which has been properly digitised (a) and analysed (b)
by the Sperm Class Analyzer
Vet. Med. – Czech, 50, 2005 (1): 24–32
and Cochran, respectively. For data that adjusted to
a normal distribution, one way ANOVA producing
significant F-values was followed by Tukey test for
multiple comparisons. For data that did not adjust
to a normal distribution, the Kruskal-Wallis nonparametric test was used followed by the MannWhitney U-test.
The semen parameters of the ejaculates used in
the present study were within the physiological
values for fertile adult stallions: a mean gel-free
volume of 43 ml, 169 million sperm/ml for sperm
concentration, 77% for sperm motility and 72% for
normal sperm morphology (estimated by subjective analysis).
Effect of the staining technique on the accuracy of image processing. Of the 3 700 spermatozoa
captured, 2 420 were correctly analysed using the
three staining procedures (Table 1). No significant
Original Paper
differences were found between the DQ (55.27%)
and HC (61.76%) staining techniques. However, HH
was by far the most accurate method (P < 0.05) with
80.47% of correctly analysed spermatozoa. The coefficients of variation obtained with HH were lower
than those obtained with DQ and HC.
Effect of the staining technique on sperm
morphometry. The morphometric values for the
sperm head and midpiece are shown in Table 2
according to the three staining procedures. Sperm
morphometric parameters were influenced by the
staining method. The DQ and HC staining methods
obtained significantly increased sperm dimensions
than HH (P < 0.05).
Effect of the number of spermatozoa analysed.
No Statistical differences were found among the subsets of 100, 150, 175 and 200 sperm cells for any of
the sperm head or midpiece morphometric parameters of the spermatozoa stained with HH (Table 3).
It suggests that the analysis of 100 spermatozoa is
sufficient for the morphometric characterization of
stallion semen sample under these conditions.
Figure 2. Morphometric parameters examined in this study (modified from Soler et al., 2003)
The morphometric parameters described for the sperm head are as follows L = Length (along the main axis), W = Width
(along the smaller axis), A = Area, P = Perimeter .Derived parameters were automatically calculated for head shape: Ellipticity (L/W), Rugosity (4πA/P2), Elongation (L – W)/(L + W), Regularity (πLW/4A). The morphometric parameters described
for the midpiece are as follows: w = width (at the intersection of the midpiece with the sperm head), d = distance (between
the main axis of the sperm head and the intersection with the midpiece), 0 = angle (formed by the axis of the midpiece and
the main axis of the sperm head), and a = area (the area occupied by the entire midpiece)
Original Paper
Vet. Med. – Czech, 50, 2005 (1): 24–32
Table 1. Percentage of correctly analysed spermatozoa (n = 2 420) from each animal stained with Diff-Quik, Hemacolor and Harris’ Haematoxylin
Staining Method (% correctly analysed)
Harris’ Haematoxylin
The superscripts indicate significant differences (P < 0.05)
CV = coefficient of variation (%)
Table 2. Effect of the three different staining procedures on morphometric parameters of the sperm head and
midpiece for all six animals
Harris’ Haematoxylin
1 205
Length (µm)
5.87 ± 0.39a
5.90± 0.41a
5.67 ± 0.36b
Width (µm)
3.07 ± 0.27a
2.97 ± 0.30b
2.85 ± 0.31c
13.42 ± 1.72c
Head Parameters
Area (µm )
14.72 ± 1.72
Perimeter (µm)
15.64 ± 0.92a
15.61 ± 1.00a
15.00 ± 0.89b
1.92 ± 0.18a
2.00 ± 0.19b
2.00 ± 0.20b
0.76 ± 0.04c
14.29 ± 1.85
0.75 ± 0.04
0.73 ± 0.04
0.31 ± 0.04a
0.33 ± 0.04b
0.33 ± 0.05b
0.96 ± 0.03a
0.96 ± 0.03a
0.95 ± 0.03b
Width (µm)
0.96 ± 0.23a
0.82 ± 0.23b
0.81 ± 0.27b
Area (µm2)
2.09 ± 0.50a
1.72 ± 0.48b
1.63 ± 0.56c
Distance (µm)
0.27 ± 0.13a
0.26 ± 0.13a
0.26 ± 0.12a
Midpiece Parameters
Angle (°)
5.63 ± 6.02
6.83 ± 7.76
6.28 ± 6.34a,b
Values are mean ± standard deviation; n = number of spermatozoa analysed; different superscripts indicate significant differences (P < 0.05)
Ellipticity = L/W; Rugosity = 4πA/P2; Elongation = (L – W)/(L + W); Regularity = πLW/4A; L = Head Length; W = Head
Width; A = Head Area; P = Head Perimeter
The use of SCA has been previously standardized
in humans (de Monserrat et al., 1995) and other
species (Gago et al., 1998; Buendia et al., 2002) with
intrinsically low coefficients of variation that demonstrate the precision and accuracy of the system,
as well as its high repeatability as no differences
are found when analyzing the same sample several
times. To apply the precision and repeatability of
Vet. Med. – Czech, 50, 2005 (1): 24–32
Original Paper
Table 3. Comparison of morphometric parameters between different sample sizes from spermatozoa stained with
Harris´ Haematoxylin for all six animals
Spermatozoa sampled
Head Parameters
Area (µm2)
Length (µm)
Width (µm)
5.64 ± 0.01
2.84 ± 0.01
13.33 ± 0.07
14.95 ± 0.04
5.66 ± 0.01
2.86 ± 0.01
13.42 ± 0.06
15.00 ± 0.03
5.66 ± 0.01
2.86 ± 0.01
13.44 ± 0.05
15.01 ± 0.03
5.66 ± 0.01
2.85 ± 0.01
13.42 ± 0.05
15.00 ± 0.03
2.00 ± 0.01
0.75 ± 0.001
0.33 ± 0.002
0.95 ± 0.001
2.00 ± 0.01
0.75 ± 0.001
0.33 ± 0.002
0.95 ± 0.001
2.00 ± 0.01
0.75 ± 0.001
0.33 ± 0.002
0.95 ± 0.001
2.00 ± 0.01
0.75 ± 0.001
0.33 ± 0.001
0.95 ± 0.001
Spermatozoa sampled
Perimeter (µm)
Midpiece Parameters
Width (µm)
Area (µm2)
Distance (µm)
Angle (°)
0.81 ± 0.01
1.61 ± 0.02
0.25 ± 0.005
6.22 ± 0.26
0.81 ± 0.01
1.63 ± 0.02
0.26 ± 0.004
6.23 ± 0.21
0.81 ± 0.01
1.63 ± 0.02
0.26 ± 0.004
6.28 ± 0.19
0.81 ± 0.01
1.63 ± 0.02
0.26 ± 0.004
6.28 ± 0.18
Values are mean ± standard error; no significant sample size effects were found
Ellipticity = L/W; Rugosity = 4πA/P2; Elongation = (L – W)/(L + W); Regularity = πLW/4A; L = Head Length; W = Head
Width; A = Head Area; P = Head Perimeter
this technology to animal species, species-specific
methods for sample preparation and staining are
needed (Davis and Gravance, 1993; Boersma et al.,
In accordance with previous studies on equine
species regarding the most suitable method of
sample preparation in this type of analysis (Davis
et al., 1993), we have used washed samples. The
performance of the SCA has been evaluated using
three staining methods. Among the methods tested
for stallions, Trypan blue and Giemsa (Kusunoki et
al., 1988), Papanicolau (Hafez, 1987) and Spermac
(Oettle, 1986) are not suitable for ASMA systems
as they result in poorly stained cells, which do not
permit digitisation (Gravance et al., 1995). The
staining methods compared in this study (DQ, HC,
HH) were chosen based on the positive results obtained in humans and in several animal species for
different ASMA systems (Lacquet et al., 1996; Gago
et al., 1998; Soler et al., 2000).
According to the results, all three staining procedures permitted the digitisation of stallion spermatozoa, although some differences are seen in
the number of recognition and digitisation errors,
showing the best results with the use of HH stain
technique. The criteria followed in this study to
evaluate the accuracy of the staining technique on
image processing included the percentage of correctly analysed spermatozoa with the three staining techniques, and the coefficients of variation
obtained with each procedure (Sancho et al., 1998).
The SCA analyzes the images captured by creating
a boundary that matches the external outline of the
microscopic image of the spermatozoa and delineating the sperm head and midpiece. In order to
obtain a correct image, the spermatozoa must contrast with the sample preparation background and
there can be no particles that interfere in the delineation of the sperm cells. The accuracy of SCA in
capturing and segmenting the spermatozoa stained
with HH was higher than those stained with DQ
and HC. These findings resemble those obtained
by Gago et al. (1998) in the cynomolgus monkey.
Spermatozoa stained with DQ and HC obtain more
intense grey-level values, thus enhancing the contrast of images. However, the same thing occurs
with other particles found in the sample, making it
necessary to eliminate a larger number of cells, con29
Original Paper
sequently slowing down the process. On the other
hand, the HH staining technique obtains suitable
grey-level values for the correct digitisation of both
sperm heads and midpieces, reduces the number of
stained foreign particles and the boundaries correctly delineate the original microscopic image.
This is supported by the fact that the coefficients
of variation calculated for each animal were lower
with HH, thus demonstrating the lower variability
and higher precision of this staining method.
Morphometric values obtained with the SCA system was affected by the staining method used and
should be interpreted accordingly. DQ and HC provide more intense grey-level values, resulting in enlarged cells, which influence the size morphometric
parameters as length, width, area and perimeter of
the head and the width and area of the midpiece. In
general terms, the relationship between the three
staining techniques for the sperm dimensions can
be described as follows: DQ > HC > HH. The impact of the staining procedure on sperm dimensions had also been tested by comparing these three
staining techniques in human (Soler et al., 2003)
and monkey spermatozoa (Gago et al., 1998). These
morphometric results are in accordance with other
authors who have found increased dimensions in
semen samples stained with DQ as compared to
Papanicolau (Menkveld et al., 1990; Gago et al.,
1998). In his study with the cynomolgus monkey,
Gago et al. (1998) found intermediate values for
HH, which were lower than HC when comparing
the DQ, HC and HH techniques. The previous results resemble those found in this study, although
we increased the HC staining time to enhance the
intensity and contrast of the images. As observed
with DQ, this process consequently increased the
sperm dimensions.
The size of the sample is also an important factor to take into consideration. We expected that a
higher numbers of spermatozoa analysed achieving a more accurate assessment of sperm morphometry. However, the results indicate that 100
properly digitised sperm cells appeared sufficient
for the morphometric characterization of a stallion semen sample under these conditions, as it
produced similar measurements as analysing 150,
175 or 200 spermatozoa. The analysis of 100 spermatozoa obtain accurate measurements and greatly
reduces the time to perform an analysis, which
was in agreement with results obtained in dog
(Rijsselaere et al., 2004) and goat (Gravance et al.,
1995). However, because heterogeneous abnormal
Vet. Med. – Czech, 50, 2005 (1): 24–32
sperm head morphology of equine species had been
described previously, it is possible that to overcome
possible problems associated with the evaluation of
infertile samples, in these semen specimens a high
number of spermatozoa should be analysed. The
animals used in the present study were considered
to be fertile on the basis of their use for breeding. Whether infertile samples are associated with
larger number of spermatozoa analysed warrants
further investigations.
In conclusion, the morphometric analysis of
the stallion spermatozoa was influenced by the
staining procedure. Harris’ Haematoxylin could
be considered the most accurate staining method
with the SCA, based on the greater percentage of
analysable cell. 100 properly digitised spermatozoa
per slide should be analysed to morphometrically
characterize the whole population in a stallion
semen sample. In short, to obtain and objective
and accurate evaluation of stallion sperm heads
and midpieces with SCA, the analysis of 100 spermatozoa per slide is recommended in samples,
which have been previously stained with Harris’
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Received: 04–11–05
Accepted after corrections: 04–12–21
Corresponding Author
Manuel Hidalgo Prieto, MV, PhD, Animal Medicine and Surgery Department, Veterinary Faculty, University
of Cordoba, Campus de Rabanales, 14014 Cordoba, Spain
Tel. +34 957 218 716, fax: +34 957 211 093, e-mail: [email protected]